Adeno-associated virus (AAV) is a replication-deficient parvovirus, the genome of which is about 4.6 kb in length, including 145 bp inverted terminal repeats (ITRs). Two open reading frames encode a series of rep and cap polypeptides. Rep polypeptides (rep78, rep68, rep62 and rep40) are involved in replication, rescue and integration of the AAV genome. The cap proteins (VP1, VP2 and VP3) form the virion capsid. Flanking the rep and cap open reading frames at the 5xe2x80x2 and 3xe2x80x2 ends are the 145 bp ITRs, the first 125 bp of which are capable of forming Y- or T-shaped duplex structures. Of importance for the development of AAV vectors, the entire rep and cap domains can be excised and replaced with a therapeutic or reporter transgene [B. J. Carter, in xe2x80x9cHandbook of Parvovirusesxe2x80x9d, ed., P. Tijsser, CRC Press, pp.155-168 (1990)]. It has been shown that the ITRs represent the minimal sequence required for replication, rescue, packaging, and integration of the AAV genome.
When this nonpathogenic human virus infects a human cell, the viral genome integrates into chromosome 19 resulting in latent infection of the cell. Production of infectious virus and replication of the virus does not occur unless the cell is coinfected with a lytic helper virus, such as adenovirus or herpesvirus. Upon infection with a helper virus, the AAV provirus is rescued and amplified, and both AAV and helper virus are produced. The infecting parental ssDNA is expanded to duplex replicating form (RF) DNAs in a rep dependent manner. The rescued AAV genomes are packaged into preformed protein capsids (icosahedral symmetry approximately 20 nm in diameter) and released as infectious virions that have packaged either + or xe2x88x92 ss DNA genomes following cell lysis.
AAV possesses unique features that make it attractive as a vector for delivering foreign DNA to cells. Various groups have studied the potential use of AAV in the treatment of disease states; however, progress towards establishing AAV as a transducing vector for gene therapy has been slow for a variety of reasons. One obstacle to the use of AAV for delivery of DNA is lack of highly efficient schemes for encapsidation of recombinant genomes and production of infectious virions [See, R. Kotin, Hum. Gene Ther., 5:793-801 (1994)].
One proposed solution involves transfecting the recombinant adeno-associated virus (rAAV) containing the transgene into host cells followed by co-infection with wild-type AAV and adenovirus. However, this method leads to unacceptably high levels of wild-type AAV. Incubation of cells with rAAV in the absence of contaminating wild-type AAV or helper adenovirus is associated with little recombinant gene expression. In the absence of rep, integration is inefficient and not directed to chromosome 19.
A widely recognized means for manufacturing transducing AAV virions entails co-transfection with two different, yet complementing plasmids. One of these contains the therapeutic or reporter transgene sandwiched between the two cis acting AAV ITRs. The AAV components that are needed for rescue and subsequent packaging of progeny recombinant genomes are provided in trans by a second plasmid encoding the viral open reading frames for rep and cap proteins. However, both rep and cap are toxic to the host cells. This toxicity has been the major source of difficulty in providing these genes in trans for the construction of a useful rAAV gene therapy vector.
Other methods have been proposed to enable high titer production of rAAV. For example, U.S. Pat. No. 5,658,776 refers to packaging systems and processes for packaging AAV vectors that replace the AAV P5 promoter with a heterologous promoter. Alternatively, U.S. Pat. No. 5,622,856 refers to constructs and methods for AAV vector production, which provide constructs formed by moving the homologous P5 promoter to a position 3xe2x80x2 to the rep genes, and optionally flanking the rep-cap and repositioned P5 promoter with FRT sequences.
There remains a need in the art for additional methods permitting the efficient production of AAV and recombinant AAV viruses for use in research and therapy.
The present invention provides novel methods, host cells, and vector constructs which permit efficient production of rAAV, by decreasing the expression of the rep78/rep68 gene products, while leaving the expression of rep52, rep40and AAV structural proteins at a normal level.
In one aspect, the invention provides a host cell containing
(a) a first nucleic acid molecule comprising from 5xe2x80x2 to 3xe2x80x2, a parvovirus P5 promoter, a spacer, an AAV rep sequence and an AAV cap sequence, wherein the spacer is of sufficient size to reduce expression of the rep78 and rep68 gene products;
(b) a second nucleic acid molecule comprising a minigene comprising a transgene flanked by AAV inverse terminal repeats (ITRs) and under the control of regulatory sequences directing expression thereof in a host cell; and
(c) helper functions essential to the replication and packaging of rAAV.
In another aspect, the invention provides a nucleic acid molecule useful in the production of recombinant AAV comprising from 5xe2x80x2 to 3xe2x80x2, a homologous P5 promoter, a spacer, an AAV rep sequence and an AAV cap sequence, wherein the spacer is of sufficient size to reduce, but not eliminate, expression of the rep78 and rep68 gene products.
In yet a further aspect, the invention provides a method for increasing the production of recombinant adeno-associated virus (rAAV) by culturing a host cell as described above, by which the rep78/rep68 gene products are reduced in expression, and isolating from the cell lysate or cell culture, high levels of recombinant AAV capable of expressing said transgene.
Other aspects and advantages of the present invention are described further in the following detailed description of the preferred embodiments thereof.